Adult I. scapularis females were fed on uninfected and A. phagocytophilum (NY18)-infected sheep as described before66 (link). Infected and uninfected female fed ticks were fixed, embedded in paraffin, and sections (4 μm) prepared and mounted on glass slides as previously described36 (link). The slides were processed36 (link) and then incubated for 14 h at 4 °C with anti-ubiquinol-cytochrome c reductase core protein I antibodies (ab96333; Abcam, Cambridge, UK) diluted 1:100 in 3% BSA/PBS and after 3 washes in PBS, developed for 1 h with either goat-anti-rabbit IgG conjugated with phycoerythrin (PE) (Sigma-Aldrich) 1:50 dilution in 3% BSA/PBS or goat anti-rabbit IgG conjugated with FITC (Sigma-Aldrich) 1:200 dilution in 3% BSA/PBS. Finally, the slides were mounted in ProLong Antifade with DAPI reagent (Molecular Probes) and examined using a Zeiss LSM 800 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with x40 oil immersion objectives.
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